Low oxygen culture conditions for maintaining retinal progenitor cell multipotency

ABSTRACT

The present invention relates to methods for culturing human retinal progenitor cells under low oxygen conditions to allow the cells to retain the ability to differentiate into photoreceptors following transplantation. The described methods provide cells that can treat a number of ocular diseases, including retinitis pigmentosa and age-related macular degeneration.

BACKGROUND OF THE INVENTION

The human retina is part of the central nervous system and, bothdevelopmentally and phenotypically, the retina shares the recalcitranceof brain and spinal cord with respect to functional repair. This isunfortunate in that, among heritable conditions alone, there are manyexamples of diseases involving the loss of retinal neurons. For example,retinitis pigmentosa, age-related macular degeneration and diabeticretinopathy are diseases characterized by the progressive death of lightsensing photoreceptor cells of the retina. These diseases are theleading causes of incurable blindness in the western world, and areincreasing rapidly in the developing Eastern world.

Since the intrinsic regenerative capacity of the human retina isextremely limited, a promising potential therapy for these diseasescurrently in research is a focus on cellular replacement. One strategyfor replacing these cells has been to transplant retinal tissue fromhealthy donors to the retina of the diseased host. While the results ofsuch studies have been encouraging in terms of graft survival, theproblem of integration between graft and host has proved daunting.Laboratory studies have focused on multipotent stem cells (alsovariously referred to as progenitor cells, immature cells,undifferentiated cells or proliferative cells) for transplantation anddifferentiation. Proliferative stem or progenitor cells have beenisolated from the hippocampus in laboratory animals, cultured andtransplanted into various sites within the central nervous system (CNS)to subsequently differentiate into neurons and glial cells. Similarly,adult hippocampal cells have been shown to be capable of migrating into,and differentiating within, the mature dystrophic retina.

The isolation of true stem cells from the neuroretina, particularlycells able to differentiate into functional photoreceptor cells both invitro and in vivo, has proven elusive. Putative retinal stem cellsderived from the ciliary marginal zone pigment epithelial layer aredescribed in U.S. Pat. No. 6,117,675. While these cells are said to becapable of proliferating in the absence of growth factor, there is noevidence that these cells are capable of integrating into a host retinaand differentiating into functional mature cells in vivo.

Commonly assigned U.S. Pat. No. 7,514,259 is directed toneuroretina-derived photoreceptor cells which are capable ofrepopulating a human retina. These cells are derived from neural retinaltissue by removing the ciliary marginal zone and the optic nerve toeliminate contamination, and can be obtained from post-natal tissue.

Apart from difficulties involving the identification of viable humanretinal progenitor cells, there are significant limitations involvingthe ability to culture these cells. Although such cells posses thecapacity to survive, to differentiate into retinal neurons, and tointegrate within the dystrophic host retina following transplantation,these cells have limited proliferative capacity. This represents a cleardistinction between human retinal progenitor cells and other lessrestricted undifferentiated cell types, such as embryonic stem cells orinduced pluripotent stem cells, which may not share such limitations.

For instance, and following isolation, human retinal progenitor cellscan only be passaged a maximum number of seven (7) times in vitrowithout loss of multipotency, including the ability to proliferate invivo and to form mature retinal cell types. This greatly limits thenumber of cells that can be obtained from a single isolate, the numberof transplants that can be performed from a single cell isolation, andthe further clinical application of these cells.

Attempts to immortalize fetal human retinal cells using SV40transfection have not proven successful since the cells fail to expressthe markers of mature differentiated cells after transplantation.Similarly, other methods for culturing cells, such as the conditionalimmortalization or downregulation of pRb, as described for Muller glialcell lines expressing retinal stem cell genes, also yield cells whichfail to differentiate into photoreceptors.

A variety of stem cell types have included, inter alia, the use of lowoxygen culture conditions. See, for instance, U.S. Pat. No. 6,759,242and U.S. Pat. No. 6,610,540 which relate to the enhanced differentiationof CNS precursor cells and neural crest stem cells under low oxygenculture conditions.

In view of the aforementioned, as well as the importance of humanretinal progenitor cells for clinical evaluation and use, it willreadily be appreciated that a need exists to improve the ability of suchcells to reproduce in vitro while maintaining multipotency properties invivo. These and other objectives of the invention will be clear from thefollowing description.

SUMMARY OF THE INVENTION

The invention is directed to the use of low oxygen culture conditionsfor enhancing the expression of retinal progenitor cells in vitro, andfor maintaining the multipotency of the cells in vivo followingtransplantation into a host. The progenitor cells according to theinvention are capable of retinal-specific differentiation intophotoreceptors, and are therefore useful for the treatment of retinaldiseases upon transplantation into a diseased eye. Thus, the inventionprovides a method to obtain a population of multipotent retinalprogenitor cells in vitro suitable for in vivo transplantation into ahost recipient. In one aspect, the population of multipotent progenitorcells is substantially homogeneous, e.g. clonally expanded.

According to the invention, human retinal progenitor cells are obtainedfrom viable neuroretinal source tissue, such as the retinal neurosphere.The cells can be added to a suitable cell culture media containingnutrients, buffering agents, and at least one exogenous growth factor.

Suitable exogenous growth factors are selected from the group consistingof epidermal growth factor (EGF), basic fibroblast growth factor (bFGF),a combination of bFGF and EGF, and a combination of EGF and bFGF andplatelet-derived growth factor (PDGF), or equivalents of each thereof.

The cells are cultured under low oxygen conditions for an effectiveamount of time. By culturing under “low oxygen conditions” is meantmaintaining the oxygen concentration in the culture media at a level offrom about 1% to about 6%. An effective amount of time intends that thecells have been cultured and passaged at least 7, or alternatively atleast 8, or alternatively, at least 9, or yet further at least 10 times.In one aspect, the passaged cells are tested to verify that themultipotency of the cells is intact and are suitable for use in in vivoapplications.

Non-limiting suitable primary sources for the cells for use in thedisclosed methods include post-natal retinal tissue, includingmammalian, e.g., murine, simian, leporidae and human adult tissuesources.

The cells and populations produced by the disclosed methods havetherapeutic use and can be autologous or allogeneic to the host patientor recipient. Because the retinal progenitor cells are capable ofdifferentiating into photoreceptor cells, they are useful to replace orrepair photoreceptor tissue in a patient and, e.g., for the treatment ofdegenerative diseases of the eye such as retinitis pigmentosa,age-related macular degeneration and diabetic retinopathy.

The foregoing embodiments and aspects of the invention are illustrativeonly, and are not meant to restrict the spirit and scope of the claimedinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other advantages and features of the invention willbecome apparent upon reading the following detailed description withreference to the accompanying figures and drawings.

FIGS. 1A-1E are photomicrographs of a human fetal retina showing themorphology of the retina. FIG. 1A depicts a retina at 20 weeksgestational age, while FIGS. 1B-1E show various retina markers at 18weeks gestational age. Scale bars are 100 um.

FIGS. 2A-2F are photomicrographs showing human retinal progenitor cellsin culture expressing various progenitor and eye development markers inprimary culture 3 days after isolation. FIGS. 2G and 2H show cell clumpsand discrete cells dissociated and cultured after first passage. Scalebars are 200 um.

FIG. 3 is a graph comparing the growth kinetics for human retinalprogenitor cells for both low oxygen cell culture conditions and normaloxygen cell culture conditions.

FIGS. 4A-4D are photomicrographs and bar graphs showing proliferativemarker Ki67 and cell cycling marker CyclinD1 expression in low andregular oxygen conditions for passages 1, 3, 5, 7, 10 and 16 (only forlow oxygen). FIG. 4E is a bar graph showing cycle checkpoint protein p53expression in regular oxygen conditions. Scale bars are 50 um.

FIGS. 5A and 5B are a photomicrograph and a bar graph showing the ratioof apoptotic cells in low and regular oxygen culture conditions onpassages 1-9. Scale bar is 50 um.

FIG. 6 is a bar graph of relative telomerase activity in hRPCs obtainedfor passages 1, 3, 5, 7, 10 and 16 for low and regular oxygenconditions.

FIGS. 7A-7E are a series of bar graphs showing the ratio of cellsexpressing specialized photoreceptor retinal cell markers in maintenanceconditions and at 2, 5 and 9 days post-differentiation on passages 1, 5,10 and 16.

FIGS. 8A-8F are a series of bar graphs showing the ratio of cellsexpressing specialized retinal cells markers in maintenance conditionsand at passages 1, 5, 10 and 16.

DETAILED DESCRIPTION OF EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All technical and patentpublications cited herein are incorporated herein by reference in theirentirety. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of tissue culture, immunology,molecular biology, microbiology, cell biology and recombinant DNA, whichare within the skill of the art. See, e.g., Sambrook and Russell eds.(2001) Molecular Cloning: A Laboratory Manual, 3^(rd) edition; theseries Ausubel et al. eds. (2007) Current Protocols in MolecularBiology; the series Methods in Enzymology (Academic Press, Inc., N.Y.);MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press atOxford University Press); MacPherson et al. (1995) PCR 2: A PracticalApproach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual;Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique,5^(th) edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization;Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds.(1984) Transcription and Translation; Immobilized Cells and Enzymes (IRLPress (1986)); Perbal (1984) A Practical Guide to Molecular Cloning;Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells(Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer andExpression in Mammalian Cells; Mayer and Walker eds. (1987)Immunochemical Methods in Cell and Molecular Biology (Academic Press,London); and Herzenberg et al. eds (1996) Weir's Handbook ofExperimental Immunology.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 1.0 or 0.1, as appropriate. It is tobe understood, although not always explicitly stated, that all numericaldesignations are preceded by the term “about”. It also is to beunderstood, although not always explicitly stated, that the reagentsdescribed herein are merely exemplary and that equivalents of such areknown in the art.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a pharmaceutically acceptable carrier”includes a plurality of pharmaceutically acceptable carriers, includingmixtures thereof.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination for the intended use. Thus, acomposition consisting essentially of the elements as defined hereinwould not exclude trace contaminants from the isolation and purificationmethod and pharmaceutically acceptable carriers, such as phosphatebuffered saline, preservatives, and the like. “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions of this invention.Embodiments defined by each of these transition terms are within thescope of this invention.

A “host” or “patient” of this invention is an animal such as a mammal,or a human. Non-human animals subject to diagnosis or treatment arethose in need of treatment such as for example, simians, murines, suchas, rats, mice, canines, such as dogs, leporids, such as rabbits,livestock, sport animals, and pets.

The term “isolated” means separated from constituents, cellular andotherwise, in which the cell, tissue, polynucleotide, peptide,polypeptide, protein, antibody or fragment(s) thereof, which arenormally associated in nature. For example, an isolated polynucleotideis separated from the 3′ and 5′ contiguous nucleotides with which it isnormally associated in its native or natural environment, e.g., on thechromosome. As is apparent to those of skill in the art, a non-naturallyoccurring polynucleotide, peptide, polypeptide, protein, antibody orfragment(s) thereof, does not require “isolation” to distinguish it fromits naturally occurring counterpart. An isolated cell is a cell that isseparated form tissue or cells of dissimilar phenotype or genotype.

As used herein, “stem cell” defines a cell with the ability to dividefor indefinite periods in culture and give rise to specialized cells. Atthis time and for convenience, stem cells are categorized as somatic(adult) or embryonic. A somatic stem cell is an undifferentiated cellfound in a differentiated tissue that can renew itself (clonal) and(with certain limitations) differentiate to yield all the specializedcell types of the tissue from which it originated. An embryonic stemcell is a primitive (undifferentiated) cell from the embryo that has thepotential to become a wide variety of specialized cell types. Anembryonic stem cell is one that has been cultured under in vitroconditions that allow proliferation without differentiation for monthsto years. Pluripotent embryonic stem cells can be distinguished fromother types of cells by the use of marker including, but not limited to,Oct-4, alkaline phosphatase, CD30, TDGF-1, GCTM-2, Genesis, Germ cellnuclear factor, SSEA1, SSEA3, and SSEA4. The term “stem cell” alsoincludes “dedifferentiated” stem cells, an example of which is a somaticcell which is directly converted to a stem cell, i.e. reprogrammed. Aclone is a line of cells that is genetically identical to theoriginating cell; in this case, a stem cell.

The term “propagate” means to grow or alter the phenotype of a cell orpopulation of cells. The term “growing” or “expanding” refers to theproliferation of cells in the presence of supporting media, nutrients,growth factors, support cells, or any chemical or biological compoundnecessary for obtaining the desired number of cells or cell type. In oneembodiment, the growing of cells results in the regeneration of tissue.In yet another embodiment, the tissue is comprised of cardiomyocytes.

The term “culturing” refers to the in vitro propagation of cells ororganisms on or in media of various kinds. It is understood that thedescendants of a cell grown in culture may not be completely identical(i.e., morphologically, genetically, or phenotypically) to the parentcell. By “expanded” is meant any proliferation or division of cells.“Clonal proliferation” refers to the growth of a population of cells bythe continuous division of single cells into two identical daughtercells and/or population of identical cells.

As used herein, the “lineage” of a cell defines the heredity of thecell, i.e. its predecessors and progeny. The lineage of a cell placesthe cell within a hereditary scheme of development and differentiation.

“Differentiation” describes the process whereby an unspecialized cellacquires the features of a specialized cell such as a heart, liver, ormuscle cell. “Directed differentiation” refers to the manipulation ofstem cell culture conditions to induce differentiation into a particularcell type or phenotype. “Dedifferentiated” defines a cell that revertsto a less committed position within the lineage of a cell. As usedherein, the term “differentiates or differentiated” defines a cell thattakes on a more committed (“differentiated”) position within the lineageof a cell.

“Retinal progenitor cells”, or “neuroretina-derived retinal stem cells”,or “retinal stem cells”, as those terms are used herein, are synonymousand mean isolated viable stem cells derived from neuroretinal tissue,such as the retinal neurosphere. The point of origin of these cells isone factor that distinguishes them from non-neural retinal cells, suchas pigmented cells of the retinal pigment epithelium, the ciliary bodyor the iris. The cells of the invention are further distinguished by aninability to proliferate in the absence of growth factors. The cells ofthe invention can derived from either pre-natal or post-natal sources,and are capable of self-renewal, multipotency, and retina-specificdifferentiation into photoreceptors. Such cells are more particularlydescribed in U.S. Pat. No. 7,514,259, the disclosure of which isincorporated by reference herein in its entirety. The retinal stem cellsof the invention are capable of: (a) self-renewal in vitro; (b)differentiating into neurons and astrocytes (but not oligodendrocytes);(c) integrating into the neuroretina following transplantation to theposterior segment of the eye; and (d) differentiation into photoreceptorcells when grafted onto a retinal explant, or into the mature eye of arecipient.

As used herein in connection with the retinal progenitor cells of theinvention, the term “multipotency”, means the ability of the retinalprogenitor cells to proliferate and form mature retinal cell types,particularly photoreceptor cells.

“Substantially homogeneous” describes a population of cells in whichmore than about 50%, or alternatively more than about 60%, oralternatively more than 70%, or alternatively more than 75%, oralternatively more than 80%, or alternatively more than 85%, oralternatively more than 90%, or alternatively, more than 95%, of thecells are of the same or similar phenotype. Phenotype can be determinedby a pre-selected cell surface marker or other marker, e.g. myosin oractin or the expression of a gene or protein,

A “biocompatible scaffold” refers to a scaffold or matrix fortissue-engineering purposes with the ability to perform as a substratethat will support the appropriate cellular activity to generate thedesired tissue, including the facilitation of molecular and mechanicalsignaling systems, without eliciting any undesirable effect in thosecells or inducing any undesirable local or systemic responses in theeventual host. In other embodiments, a biocompatible scaffold is aprecursor to an implantable device which has the ability to perform itsintended function, with the desired degree of incorporation in the host,without eliciting an undesirable local or systemic effects in the host.Biocompatible scaffolds are described in U.S. Pat. No. 6,638,369.

As used herein, the terms “treating,” “treatment” and the like are usedherein to mean obtaining a desired pharmacologic and/or physiologiceffect. The effect can be prophylactic in terms of completely orpartially preventing a disorder or sign or symptom thereof, and/or canbe therapeutic in terms of a partial or complete cure for a disorderand/or adverse effect attributable to the disorder. Examples of“treatment” include but are not limited to: preventing a disorder fromoccurring in a subject that may be predisposed to a disorder, but hasnot yet been diagnosed as having it; inhibiting a disorder, i.e.,arresting its development; and/or relieving or ameliorating the symptomsof disorder, e.g., macular degeneration. As is understood by thoseskilled in the art, “treatment” can include systemic amelioration of thesymptoms associated with the pathology and/or a delay in onset ofsymptoms such as chest pain. Clinical and sub-clinical evidence of“treatment” will vary with the pathology, the individual and thetreatment.

The terms “physiologic”, or “physiologic oxygen”, as used herein, referto the low oxygen concentrations of the invention of from about 1% toabout 6%, and the particularly preferred concentrations of from about 2%to about 4% as measured in the culture media. As is apparent to theskilled artisan, the oxygen level of the culturing device may beslightly higher in order to obtain the appropriate oxygen concentrationin the media to which the cells are exposed.

The oxygen concentration in mammalian cell tissues in vivo typicallyvaries from 0.5% for the retina to 19% for the upper airway epithelia.In the retina in particular, the adult retina oxygen concentration (oroxygen “tension”) varies from about 0.5% for the inner nuclear layer toabout 7% for the outer segments of the retina. For most cell cultures,the oxygen concentration is normally maintained at about 20%, theso-called “normaoxic” level. The term “anoxic”, as may be used herein,refers to oxygen concentrations of less than about 1%.

A “composition” is intended to mean a combination of active agent, cellor population of cells and another compound or composition, inert (forexample, a detectable agent or label) or active, such as a biocompatiblescaffold.

A “pharmaceutical composition” is intended to include the combination ofan active agent with a carrier, inert or active such as a biocompatiblescaffold, making the composition suitable for diagnostic or therapeuticuse in vitro, in vivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.The compositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see Martin, Remington'sPharm. Sci., 15th Ed. (Mack Publ. Co., Easton (1975)).

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations, applications or dosages.

Cells, Populations and Compositions

The invention relates to a process for producing an isolated populationof multipotent retinal stem cells in vitro suitable for therapeutic use.The method requires culturing an isolated retinal progenitor cell underlow oxygen conditions, typically wherein the oxygen concentrationutilized in the culture media is from about 1% to about 6%, preferablyfrom about 2% to about 4%. It has been found that the methods of theinvention permit the expansion of human retinal progenitor cells invitro, increase cell proliferation and multipotency marker expression,and decrease cell apoptosis. Importantly, the expanded cells of theinvention maintain the ability to differentiate into specialized retinalcells, particularly photoreceptor cells.

Thus, in one aspect, the invention provides an in vitro method forpreparing an isolated population of multipotent progenitor retinalprogenitor cells, comprising culturing an isolated primary retinalprogenitor cell under low oxygen conditions, wherein the low oxygenconditions comprise from about 1% to about 6% oxygen content of theculture medium, for an effective amount of time while maintainingmultipotency of the cells, The isolated retinal progenitor cell to becultured can be a primary retinal cell isolated from host tissue, e.g.,a living host or a cadaver, prenatal sources, fetal tissue or adulttissue, and can be isolated from the retinal neurosphere. The cells canalso be identified by markers, that include, for example, Otx2, Sox2,Pax6—eye field development transcription factors; CyclinD1, Ki67,hTERT—proliferative markers; cMyc, Klf4, Oct4—“stemness” transcriptionfactors; SSEA4—surface antigen, characteristic for undifferentiatedcells. Methods of screening for such factors are known in the art anddescribed herein.

Culturing in low oxygen can be accomplished in more than one culturemedium as described below. Prior to use, the cells can further beisolated from the medium and combined with an appropriatepharmaceutically acceptable carrier, non-limiting examples of which aredescribed herein and known to the skilled artisan. In addition and priorto use, the isolated cells or cell populations can be further assayedfor multipotency by screening, e.g., HIF1 alpha and HIF2 alpha as wellas for the above-noted stem cell markers or independently or incombination with a telomerase assay as described below.

Suitable conditions for culturing include culturing in the presence ofat least one growth factor as described herein and for at least 7, oralternatively at least 8, or further at least 9 or yet further at least10 passages in low oxygen conditions.

The primary source of the cells can be from any suitable animal speciesas described herein. In one aspect, the primary retinal progenitor cellsare isolated from post-natal retinal tissue, e.g., from the retinalneurosphere.

The expanded population of cells is further provided herein. Thus, inone aspect this disclosure provides an isolated population, e.g., anisolated substantially homogenous population of multipotent retinalcells produced by a method of one any of claims 1, 1b and 2-7.

According to the invention, isolated human retinal progenitor cells canbe derived by the dissection of the human neural retina. Duringdissection, it may necessary to manage the highly tenacious vitreous gelcomponent. This can be accomplished using a variety of techniques, aloneor in combination, including vitrectomy, ocular inversion, mechanicalresection and absorbent debridement, as well as enzymatic digestion.Suitable enzymes for this purpose include, but are not limited to,hyaluronidases and collagenases. It may also be advantageous to removenon-neural retinal tissue from the specimen used for retinal stem cellisolation. The non-neural tissue includes the optic nerve head andepithelium of the pars plana of the ciliary body, which is typicallyadherent along the peripheral margin (ora serrata). The tissue ispreferably handled using aseptic techniques.

The isolated neuroretinal tissue can be mechanically macerated, andpassed through a nylon mesh screen of about 100 micron pore size todissociate the isolated neuroretinal tissue into cells. The use of asterile small pore filter screen for the mechanical dissociation of thetissue permits the minimization of the use of enzymes that can degradecell surface molecules such as growth factor receptors.

An aliquot of cells from the dissected tissue can then be placed in aculture vessel, such as a plastic tissue culture flask, which ispreferably coated with a protein layer. Advantageously, the layer may bepolyornithine overlaid with laminin or fibronectin.

The aliquot of cells can then be incubated, if preferred, in a firstcell culture medium to provide an initial cell concentration for about24 hours at about 35° C.-39° C., in low oxygen conditions (1% to 6%,preferably 2% to 4%, and most preferably 3% in the culture media). Thefirst cell culture medium can include a physiologically balanced saltsolution containing a D-glucose content of from about 0.5-3.0 mg/liter,preferably about 1 mg/liter, N₂ Supplement, and about 5-15% fetal calfserum, as well as 5-15% by volume neural/retinal-conditioned media andan effective amount of at least one antibiotic, such as gentamycin.

After about 24 hours of incubation in the first culture medium, thatmedium can be removed from the culture vessel. Then, a second culturemedium that is essentially serum-free, is added to the culture vessel.The second culture medium can include a physiologically balanced saltsolution containing a glucose content of about 0.5-3.0 mg/liter,preferably 1 mg/liter (e.g., DMEM/F-12 high glucose), N₂ Supplement, atleast one growth factor at a concentration of about 30-50 ng/ml pergrowth factor, an effective amount of L-glutamine (about 0.5-3.0 mM,preferably about 1.0 mM), an effective amount of neural progenitorcell-conditioned medium, and an effective amount of at least oneantibiotic, such as penicillin and/or streptomycin, in a low oxygenconcentration as described previously. Advantageously, penicillin and/orstreptomycin may be added as follows: 10,000 units/ml pen, 10,000microgram/ml strep, added 1:50-150, preferably 1:100, for a finalconcentration of 100 units/ml, 100 microgram/ml, respectively, in theculture medium. Those of ordinary skill in the art reading thisspecification will appreciate that minor modifications can be made tothe design of the culture media components and operating conditions.

The cell isolation and culture method of the invention can typicallyinclude the regular removal of non-viable cells and a portion of theculture medium from the culture vessel in which the cells are cultured,and replacing said portion with an equivalent amount of fresh, secondculture medium. This culture maintenance step may be performedapproximately every 2-7 days during the lifetime of the cell culture.

Maintenance of low oxygen conditions can be achieved using commerciallyavailable incubators with oxygen concentration control, or by placingculture flasks into chambers with manually controlled or digitallycontrolled temperature settings, humidity and concentrations of oxygenand carbon dioxide.

The survival and effectiveness of human retinal progenitor cells invitro is influenced by numerous factors including the incorporation ofvarious additives in the culture medium such as supplements, mitogens,serum and growth factors. In particular, the culture medium shouldinclude an exogenous growth factor to induce proliferation and survivalin vitro. Effective exogenous growth factors include neurotrophins;mitogens; cytokines; growth factors; hormones; and combinations thereof,as will be appreciated by one of ordinary skill in the art.Advantageously, the culture medium includes one of the following growthfactors or combinations of growth factors: epidermal growth factor(EGF), basic fibroblast growth factor (bFGF), a combination of bFGF andEGF, and a combination of EGF and bFGF and platelet-derived growthfactor (PDGF) or an equivalent of each thereof.

Without being bound by theory, it has now been found that themaintenance of the cells, and the preservation of cell multipotency, isinfluenced by low oxygen concentration in the culture medium. While lowoxygen concentration affects cell metabolism by several pathways, it hasbeen found that the main mediators of cellular activity are the twoHypoxia Inducible Factors HIF1a and HIF2a (Hypoxia Inducible Factor 1alpha and 2 alpha). These mediators are constitutively expressed buthydroxylated and ubiquitinated at oxygen concentrations of more thanabout 6%. Conversely, if the oxygen level is decreased, the HIF alphasubunits dimerise with the HIF beta subunit (ARNT), and the resultingcomplex is transported to the cell nucleus where it functions as a basichelix-loop-helix transcription factor.

HIF1 alpha expression is found in all types of cells. However, HIF2alpha expression is limited to certain organs, such as the brain, heart,lung, kidney, liver, pancreas, intestine and retina. HIF1 alpharegulates metabolic pathways (shift to glycolysis), tissue remodeling(increase in metalloproteases and decrease in ECM production), migration(cheomokine expression), pro-survival pathways (for different cell typesand oxygen tension: the HIF1a-ARNT complex may cause an increase inapoptosis via BNIP3, NIX, or a decrease in apoptosis by p53 inhibitionand Epo activation), and genome methylation (3h3mCoenzA, JMJD1A). HIF2alpha activates TGF alpha, the multipotency transcription factors Oct4,cMyc, Sox2, and Cyclin (D1, D2 and E2F) expression. Both HIF1a and HIF2ainfluence growth factor signaling.

The ability to significantly expand the number of cells to produce apopulation of multipotent retinal progenitor cells from an initialisolated population limited in size is critical for clinical studiesinvolving such cells, as well as the eventual use of the cells for thetreatment of degenerative ocular diseases using transplantationtechniques. The current level of cell production achievable usingconventional techniques is on the order of 10⁹ to 10¹⁰ cells for invitro expansion of a single isolate. This level is generally inadequatefor clinical use. It has now been found that this level of proliferationcan be increased to 10¹⁸ to 10¹⁹ cells for each cell source by using lowoxygen culture conditions in vitro.

In addition, it has also been found that the use of low oxygen cultureconditions resulted in a doubling of the proliferation speed, therebydecreasing the accumulation of genome changes while promoting thepositive selection for viable progenitors. These results are confirmedby the high levels of Ki67 and Cyclin D1 expression compared to passage0 (isolation time point). The number of Ki67 expressing cells decreasedsignificantly with passage in 20% oxygen conditions, but not in 3%oxygen conditions.

As indicated previously, the presence of both HIF (1 and 2) alphaisoforms was observed in human retinal progenitor cells. However, thesemarkers play different roles in cell fate. HIF1 alpha primarily mediateshypoxic effects, while HIF2 alpha mediates the physiologic effects oflow oxygen concentration. Hypoxic effects include stabilizing p53,blocking cell cycle progression and proliferation, and activatingapoptosis. Physiologic effects include an increase in proliferation,multipotency gene expression, TERT upregulation, and cell cycleprogression. HIF2 alpha is stabilized in human retinal progenitor cellsat oxygen concentrations of 1% to 6%, and preferably from 2% to 4%, andthis stabilization is maintained during cell passaging. Moreover, thefunctional properties of human retinal progenitor cells, includingmultipotency, are also stabilized.

Under normoxic conditions, the expansion of human retinal progenitorcells in vitro is linked to a loss in the ability of the cells todifferentiate into mature cell types, such as photoreceptors andganglion cells. However, under low oxygen conditions, human retinalprogenitor cells have been shown to retain the ability to differentiateinto photoreceptors.

Summarizing, the use of low oxygen conditions for culturing humanretinal progenitor is beneficial and results in rates of proliferationdouble the rates for normoxic conditions, and further, the rates of cellproliferation are preserved at least through passage 16. For normoxicconditions, the cell proliferation rate reached a plateau at passages 5and 6, while the use of low oxygen conditions showed only a smalldecrease in the proliferation rate. Further, cells expanded under lowoxygen conditions have been shown to preserve their genomic integrityand multipotency properties. This also results in an increase in cellsurvival and integration stability following transplantation due to aswitch to glycolysis, and the activation of pro-survival pathways andmatrix metalloproteases.

Also provided by this invention are methods to genetically modify theisolated cell population by inserting or modulating the expression ofone or more genes using methods known to the skilled artisan. In oneaspect, such modification is achieved by transducing a polynucleotideencoding the gene into the source cell by any suitable method. Forexample, the polynucleotide of interest is inserted into a vector suchas a viral vector which is then contacted with the cell under conditionsthat facilitate transfer of the vector and polynucleotide into the cell.The recipient cell is grown or propagated under suitable conditions toexpress the inserted gene. In other aspects, the cell is modified toenhance expression of the endogenous gene of interest. In furtheraspects, the genes are overexpressed as compared to a wild-typecounterpart cell by inserting numerous copies of the polynucleotide oralternatively, enhancing expression of the endogenous gene of interest.Compositions and methods to reduce or block endogenous expression arealso utilized. To promote expression, polynucleotides encoding theprotein of interest can be introduced. To inhibit expression,polynucleotides or agents such as blocking antibodies, ribozymes,antisense polynucleotides or other inhibiting agents, can be introducedinto the cell or population of cells.

Therapeutic Use

This invention also provides methods for replacing or repairingphotoreceptor cells in a patient in need of this treatment comprisingadministering to the patient an effective amount of the isolatedpopulation or composition as described herein. In one aspect, thecompositions and cell populations can treat or alleviate the symptoms ofretinitis pigmentosa in a patient in need of the treatment byadministering an effective amount of the populations or compositions.Further provided is a method for treating or alleviating the symptoms ofage related macular degeneration in a patient in need of this treatment,comprising administering to the patient an effective amount of theisolated population or composition thereby treating or alleviating thesymptoms of age related macular degeneration in said patient. For all ofthese treatments, the cells can be autologous or allogeneic to thepatient. Patients include without limitation, mammals, such as murines,canines, felines, and human patients.

Administration of the cells or compositions can be effected in one dose,continuously or intermittently throughout the course of treatment.Methods of determining the most effective means and dosage ofadministration are known to those of skill in the art and will vary withthe composition used for therapy, the purpose of the therapy and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician. Suitable dosage formulations and methods of administering theagents are known in the art. In a further aspect, the cells andcomposition of the invention can be administered in combination withother treatments.

Screening Assays

The present invention provides methods for screening various agents thatmodulate the differentiation of a retinal progenitor cell. For thepurposes of this invention, an “agent” is intended to include, but notbe limited to a biological or chemical compound such as a simple orcomplex organic or inorganic molecule, a peptide, a protein (e.g.antibody), a polynucleotide (e.g. anti-sense) or a ribozyme. A vastarray of compounds can be synthesized, for example polymers, such aspolypeptides and polynucleotides, and synthetic organic compounds basedon various core structures, and these are also included in the term“agent.” In addition, various natural sources can provide compounds forscreening, such as plant or animal extracts, and the like. It should beunderstood, although not always explicitly stated, that the agent isused alone or in combination with another agent, having the same ordifferent biological activity as the agents identified by the inventivescreen.

To practice the screening method in vitro, the isolated population ofcells is obtained as described above. When the agent is a compositionother than a DNA or RNA, such as a small molecule as described above,the agent can be directly added to the cells or added to culture mediumfor addition. As is apparent to those skilled in the art, an “effective”a mount must be added which can be empirically determined. When theagent is a polynucleotide, it can be directly added by use of a gene gunor electroporation. Alternatively, it can be inserted into the cellusing a gene delivery vehicle or other method as described above.Positive and negative controls can be assayed to confirm the purportedactivity of the drug or other agent.

The invention may be further described and illustrated in the followingexamples which are not in tended to limit the scope of the inventionthereby.

EXAMPLES

Materials and Methods

Retina Morphology

The morphology of the neural retina at 20 weeks gestational age wasinvestigated. One eye cup was fixed in Karnovsky fixative, embedded inplastic and cut (2 um thick) with H&E staining One eye cup was fixed in4% PFA, embedded in OCT, cryosectioned (6 um thick) with furtherimmunohystochemistry for Ki67, Sox2, Pax6, Recoverin, beta3-tubulin,laminin, Opsin Red/Green, Opsin Blue, Rhodopsin, Calbindin and mGluR6.

Cell Isolation

hRPCs (human retinal progenitor cells) were isolated as described, withsmall modifications, in the following references: Klassen, H. J. et al.,Multipotent Retinal Progenitors Express Developmental Markers,Differentiate into Retinal Neurons, and Preserve Light-MediatedBehavior, Invest. Opthalmol. Vis. Sci., 2004, 45(11), pages 4167-4173;Klassen, H. et al., Isolation of Retinal Progenitor Cells fromPost-Mortem Human Tissue and Comparison with Autologous BrainProgenitors, J. Neuroscience Research, 2004, 77(3), pages 334-343;Klassen, H. et al., Progenitor Cells from the Porcine Neural RetinaExpress Photoreceptor Markers after Transplantation to the SubretinalSpace of Allorecipients; Stem Cells, 2007, 25(5); pages 1222-1230.Briefly, whole neuroretinas from human fetal eyes (16-20 weeksgestational age) were dissected, dissociated in 0.1% collagenase I(Sigma) during 4 cycles (1.5 hour of fermentation in total), and platedin modified Ultraculture media (10 ng/ml rhEGF, 20 ng/ml rhbFGF,Pen/strep, Nystatin and L-glutamine) or frozen. The amount and viabilityof single cells and clumps were estimated using Trypan blue and ahaemocytometer.

Cell Culture

Cells were plated at a density of approximately 10,000 cells/cm² andcultured under either physiologic oxygen (3% oxygen) or normoxic (20%oxygen) conditions at 37° C., 100% humidity, 5% CO² in modifiedUltracutlure media (10 ng/ml rhEGF, 20 ng/ml rhbFGF, Pen/strep, Nystatinand L-glutamine) on flasks coated with bovine serum fibronectin (Akron).Cells were passaged at 75%-85% confluence (usually each 2-5 days) usingTrypsin-EDTA solution. At each passage, the cells were counted andplated at the density mentioned above. Low-oxygen conditions werecreated in a Thermo 150i incubator, not exceeding the limit of 6%oxygen.

Cell Proliferation and Growth Curve

Cell proliferation in both conditions was assessed during routinepassaging by cell count via a haemocytometer (at least in two flasks foreach passage/source). CyQuant NF assay (Invitrogen) was performed toestimate proliferation speed on each passage: a calibration curve wasbuilt by plating 1000, 2000, 4000 and 8000 cells in wells of a 96-wellplate (BD Optilux). The amount of cells in experimental wells (4000cells/well) was assessed by CyQuant staining after 48 and 72 hours (n=4for calibration curve and n=6 for experimental wells).

Apoptosis

hRPCs (p1-p9) for TUNEL assay (Roche) were plated in 16-well slidescoated with fibronectin, the same way as for maintenance conditions(4,000 of alive cells in each well, hRPC media with supplements); 48hours after plating cells were fixed, permeabilised (0.01% Triton-X,0.01% sodium citrate), and stained for double-stained DNA breaks. Slideswere mounted, and a cell count was performed in 9 fields of view foreach condition. Western blot analysis for pro-survival pathway proteinsp44/42 and p38 (Cell Signaling) was performed for passages 1, 5 and 10in both conditions (protein was collected after 4 days in culture).

Immunocytochemistry

Cells were assessed via immunocytochemical analysis at passages 1, 3, 5,7 and 10 in both conditions, and on passage 16 in 3% oxygen forsternness and proliferation marker expression: Otx2, Sox2, Pax6—eyefield development transcription factors; CyclinD1, Ki67,hTERT—proliferative markers; cMyc, Klf4, Oct4—“stemness” transcriptionfactors; SSEA4—surface antigen, characteristic for undifferentiatedcells. For this purpose, 4,000 cells were plated in each well of 16-wellfibronectin coated chamber glass slides (Nunc). After 24 hours ofincubation under appropriate conditions, cells were washed in PBS, fixed(cold, freshly prepared 4% PFA), permeabilised (0.02% Triton X-100 in 5%BSA), blocked and stained with primary antibodies overnight at 4° C.,and secondary antibodies (1:50, Goat Cy3-conjugated anti-rabbit oranti-mouse, Jackson Immunoresearch) for 1 hr at room temperature.

Western Blot

hRPCs cultured under the conditions described above (3% and 20% oxygen)for 4 days were harvested for protein analysis on passages 1, 3, 5, 7,10 and 16, lysed in RIPA buffer, and analyzed for protein expression byWestern blot. Proteins were separated on 8% SDS-PAGE gel, transferred toa PVDF membrane (Bio-Rad), which was blocked with 5% non-fat milk(Bio-Rad) in TBS-T, and stained with antibodies diluted in 5% BSA inTBS-T (EGFR, HIF1alpha, HIF2alpha, hTERT, Nestin, Sox2, Oct4, Klf4,cMyc, p44/42, and p38). Resulting bands were imaged with ECL Plus(Perkin Elmer) and CL-Xposure film (Thermo Sientific). Anit-bActinHRP-linked antibodies (Abcam) were used as a loading control. Bandsquare was measured using ImageJ.

Telomerase Activity Assay

Telomerase activity was assessed in both experimental conditions onpassages 1, 3, 5, 7, 10 and 16 by the TRAPeze method according to themanufacturer's (Millipore) instructions. Briefly, cells were harvested,lysed in CHAPS buffer for 30 minutes on ice, and the telomers wereamplified for 30 minutes at 30° C. The products were amplified usingPlatinum Taq (Invitrogen), separated by PAGE gel electrophoresis(non-reducing conditions) and stained with SYBR Gold (1:10000,Invitrogen) for 20 minutes at room temperature.

Differentiation Abilities In Vitro

To assess the ability of hRPCs to differentiate in vitro, hRPCs expandedin 3% oxygen were plated from passages 1, 5, 10 and 16 on fibronectin &laminin-coated 16-well slides. The cells were cultured indifferentiating media (DMEM/F12, 1XNEAA, L-glu, 5% HI FBS, Pen/strep andNystatin) in 3% oxygen. On days 2, 5 and 9, cells were fixed and stainedfor blue opsin (short-wave cones), red/green opsin (long-wave cones),rhodopsin (rods), recoverin (photoreceptor precursor), calbindin(horizontal cells), GFAP (Muller & ganglion cells), Glutamine sythetase(ganglion cells), MAP2 and Cyclin D3 (gangion cells) and PKCa (bipolarcells). The same staining was performed for hPRC on the same passagesbut after 24 hours in maintenance conditions. The ability todifferentiate was estimated by comparing the number of cells expressingmature retinal markers in differentiating versus maintenance conditions.

Results

Eye Morphology

The human neural retina at 20 weeks gestational age is 250 um thick,compared to 500 um thick for an adult retina. Three layers of the retinacan be distinguished at this age. The ganglion cell layer is separatedfrom the others by the inner plexiform layer, while the inner and outernuclear layers only start to diverge. An outer plexiform layer is notpresent at this stage of development. Due to the absence of rods andcones outer segments, the outer limiting membrane is not present, whilethe space between the ganglion cell layer and inner limiting membrane iswider than the inner plexiform layer. See FIG. 1A, which shows that theouter and inner nuclear layers are not completely separated, with outersegments not present, while the inner limiting membrane (arrows) andganglion layer have been formed.

At 18 weeks gestational age, the innermost portion of the ganglion celllayer is presented by single, recoverin-positive cells. Signs ofdeveloped photoreceptors (staining for Blue and Red/Green opsins,rhodopsin, outer segments showed negative) have not been found, whichmakes neural retina at this age good for precursor isolation. Recoverinexpression was limited by several cell layers within the outer nuclearlayer and single cells (possibly photosensitive ganglion cells) in theganglion cell layer. Ki67 is present in the middle of the conglomerateof inner and outer nuclear layers—but not in the layers, expressingrecoverin. Sox2 is expressed in all cells within the neural retina, butat low levels in cells close to the outer limiting membrane. FIG. 1Bshows proliferative marker Ki67 present in the middle of the inner andouter nuclear layers, but not in the layers, expressing recoverin. FIG.1C shows neural progenitor marker Sox2 expressed in all cells within theneural retina, with slightly decreased levels in the outer nuclearlayer. FIG. 1D shows that recoverin (photoreceptor precursor marker)expression was limited to the outer nuclear layer and single cells inthe ganglion cell layer. FIG. 1E shows that the Pax6 marker is presentin both the outer and inner nuclear layers.

Cell Isolation and Cell Culture

It was found that hRPC isolation can be performed up to 24 hours afterenucleation. From each pair of eyes we can obtain 17.8 mln (+/−1.5 mln)single cells (with viability of 52%+/−16% mln) and 1.5 mln (+/−0.5 mln)clumps, consisting of 10-100 cells. The clump viability, indirectlyassessed by adhesion to fibronectin during the first hour after plating,was about 70% and does not vary between two conditions. Cell and clumpviability does not decrease greatly after freeze/thaw. Most of the smallsingle cells, isolated from the retina, die on the second-third dayafter plating, and most of the primary pool was obtained from the clumpsoutgrowth. FIG. 2H shows the clumps. After the first passage, cellsslightly increased in size but kept high nucleus/cytoplasm ratio, obtaintriangle or spindle morphology and the culture became more homogeneous.FIG. 2G shows dissociated and cultured cells becoming more homogeneous,obtaining higher levels of Ki67 expression, losing recoverin andincreasing in size.

The ratio of Ki67 and Pax6 expressing cells in culture was approximatelythe same as in the retina prior to isolation, but much lower than duringfurther passaging. The ratio of recoverin-positive cells was decreased,and the cells did not survive further passaging, while the rate of Sox2expression increased, which suggests a sort of positive selection forprogenitors (Sox2) and a negative selection for late precursors(recoverin). Despite some reports that cell adhesion decreases in lowoxygen conditions due to Integrin downregulation, no differences wereobserved in hRPC adhesion to fibronectin-coated (75 ug/ml) culturesurfaces. As shown earlier, proper adhesion is critical for survival andexpansion of these cells. In specified maintenance conditions, hRPCcells remain unchanged until passages 3-4 in regular oxygen conditions,and passages 9-10 in low oxygen conditions, while their morphologyflattened. FIG. 2A shows that Ki67 expression in primary culture was lowcompared to further passages. FIGS. 2B and 2C show that Pax6 and Otx2were expressed only in come cells, while FIG. 2D shows that recoverinwas expressed in some positive cells. FIG. 2F shows that Sox2 wasexpressed in most of the cultured cells.

Cell Proliferation and Growth Curve

The rate of hRPC proliferation in 20% oxygen was constant during thefirst 4-5 passages (depending on the cell source), reaching a plateau atpassages 5-6 (about 20 days in culture). This data is shown in FIG. 1which graphs the growth kinetics of human retinal progenitor cells asthe number of cell divisions vs. time (days) for 3% oxygen and 20%oxygen. After “exiting” the plateau, which took about 10 days, thegrowth rate in 20% oxygen decreased compared to earlier passages. A“negative gain” in CyQuant Assay on passages 8-9 in 20% oxygen can beexplained by the increase in the apoptosis level. In 3% oxygen, a slightdecrease in the proliferation rate after the first 5 passages (12 daysin culture) was observed, but the population double time remains at alevel lower than 1.5 days up to and including passage 16 (the point atwhich the experiment ended).

FIG. 3 is a graph showing the growth kinetics of human progenitor cells.The estimated size of the hRPC population, expanded in both low andregular oxygen conditions, is established for each of the 8 differentsources. The points on the graph represent the passaging procedure whencells were counted (at least twice for each source/passage).

Proliferative Markers

The observed increase in hRPC proliferation in 3% oxygen conditionscorrelates with the increase in expression of markers Ki67 and CyclinD1.An increase in p53 expression at all passages in normoxia conditionscompared to hypoxia conditions was also observed. See FIGS. 4A-4D whichdepict photomicrographs and bar graphs for proliferative marker Ki67 andcell cycling marker CyclinD1 expression in low and regular oxygenconditions for passages 1, 3, 5, 7, 10 and 16 (only for low oxygen).FIG. 4E is a bar graph showing that cycle checkpoint protein p53expression is higher in regular oxygen conditions.

Apoptosis

The increase in p53 expression together with growth kinetics on passages8 and 9 is linked with the higher apoptosis levels in 20% oxygenconditions as determined by the TUNEL assay. It does not exceed the 2%level in the hypoxia group at passages 1-7, and increased to 5% atpassage 8. In the normoxia group, it increased from the 2% level onpassage 1 to 6% on passages 5, 6, and 7, and on passage 8 it reached20%. The variability between cell sources in the number ofTUNEL-positive cells was also higher in the normoxia group. The“pro-survival” effect of hypoxia was also supported by the observationthat after trypsinization and freeze/thawing hRPCs in 3% oxygenconditions havw higher vialbility (less floating cells). The same lowoxygen anti-apoptotic protection was shown in vitro and in vivo indifferent cell types via activation of pro-survival pathways.

FIGS. 5A and 5B are a photomicrograph and a bar graph showing the ratioof apoptotic cells in low and regular oxygen culture conditions onpassages 1-9. Although not wishing to be bound to any specificexplanation or theory of operability, a possible mechanism for thisresult is the upregulation of p39 and/or p44/42 in 3% oxygen.

Telomerase Expression and Activity

It has been shown that hRPCs express hTERT both in 3% and 20% conditionson all passages. The TRAPEZE assay has shown that telomerase activity isdecreased with passage in both conditions but is higher in 3% vs. 20%oxygen conditions, and is preserved until passage 16 in low oxygen,possibly allowing retinal progenitors to divide without telomersshortening. See FIG. 6 which is a bar graph of the relative telomeraseactivity in hRPCs obtained for passages 1, 3, 5, 7, 10 and 16 for lowand regular oxygen conditions. The telomerase products wereapproximately 50 bp.

Multipotency Marker

ICC and Western Blot have shown that culturing in 3% oxygen tensionstabilizes HIF2 alpha, increases HIF1 alpha stabilization, andupregulates cMyc, KLF4, Oct4 and Sox2. It has also been shown that theexpression of specific eye field development transcription factors(Pax6, Sox2, Otx2) and Nestin is maintained up to passage 16 in 3%oxygen conditions and up to passage 10 in 20% oxygen conditions, whichsuggests that they are not key factors in hRPC expansion in bothconditions. Sox 2 was upregulated in 3% oxygen conditions, possible dueto HIF2a activity. The same upregulation was observed for cytoplasmaticOct4 (52 kDa, expressed perinuclearily), also due to HIF2astabilization. Despite the fact that Pax6 and Otx2 expression wasobserved at all passages in maintenance conditions, the pattern changedduring differentiation. Instead of equal expression (the same intensity)in all nuclei, a dominant subpopulation with upregulated Pax6 appeared.Otx2 expression shifted from whole cell (nucleus and cytoplasm) tonucleus only, but it was still expressed in most cells. Sox2 expressiondecreased during differentiation. SSEA4 upregulation in low oxygenconditions, shown for MIAMI and hESC cells, holds for hRPC cells onlyafter passage 3. During isolation and on passage 1, the number of SSEA4expressing cells was lower in 3% oxygen conditions, but increased onpassage 3 and later passages.

Differentiation

The main characteristics of hRPC cells are functional—the ability todifferentiate into specialized retinal cells. The ability of hRPC's(passage 3) to generate about 35% recoverin expressing cells, 7%blue-opsin expressing cells, and 15% of rhodopsin expressing cells afterexpansion in 20% oxygen tension and 7 days in differentiating conditions(media supplemented with 5% FBS without mitogens) was previouslyobserved. Under the same conditions, recoverin, CRX or opsin-positivecells were not detected after differentiation of passage 6 hRPCs. Duringcell expansion under 3% oxygen conditions, it has been shown thatculturing the cells under low oxygen tension in maintenance conditionsdoes not drive spontaneous differentiation of human retinal progenitorcells into rods, cones, ganglion, bipolar or glial cells (less than 0.5%in maintenance conditions according to ICC staining) See FIGS. 7 and 8.

Summarizing the results of the above experiments, hRPCs expanded in 3%oxygen are able to generate specialized retinal cells (photoreceptors),including rods and cones. On passage 1 compared to other passages (5,10, 16), higher amounts of specialized cells were observed afterdifferentiation. hRCPs on passage 1 tended to form more specializedcells on day 2 as compared to other passages, which suggests that thecell culture on early passage is rich with “easy differentiatingprecursors” that are lost during expansion. The ability of hRPCs togenerate specialized retinal cells is greatly decreased on otherpassages as compared to passage 1, but is relatively constant betweenlater passages. This suggests that progenitors cultured in 3% oxygenunder maintenance conditions do not lose the ability to generatephotoreceptors, ganglion or bipolar cells. On later passages, lessphotoreceptors were generated, but more bipolar and glial cells. Duringdifferentiation, the downregulation of Pax6 (according to ICC staining,cells continue to express this marker, but at lower levels) and Sox2(the staining pattern switches from nuclear to perinuclear andcytoplasmic) was observed.

The human retinal progenitor cells of the invention may be used forstudying the development of the retina and eye, as well as factorsaffecting such development, whether beneficially or adversely. ThesehRPCs can also be used for clinical trials by transplantation into asuffering from dysfunctions of the eye. They may be used advantageouslyto repopulate or to rescue a dystrophic ocular tissue, particularly adysfunctional retina. Retinal dysfunction encompasses any lack or lossof normal retinal function, whether due to disease, mechanical orchemical injury, or a degenerative or pathological process involving therecipient's retina. The hRPCs may be injected or otherwise placed in aretinal site, the subretinal space, vitreal cavity, or the optic nerve,according to techniques known in the art. This includes the use ofbiodegradable substrates as a carrier for the hRPCs.

Advantageously, the hRPCs of the invention may be used to compensate fora lack or diminution of photoreceptor cell function. Examples of retinaldysfunction that can be treated by the retinal stem cell populations andmethods of the invention include but are not limited to: photoreceptordegeneration (as occurs in, e.g., retinitis pigmentosa, conedystrophies, cone-rod and/or rod-cone dystrophies, and maculardegeneration); retina detachment and retinal trauma; photic lesionscaused by laser or sunlight; a macular hole; a macular edema; nightblindness and color blindness; ischemic retinopathy as caused bydiabetes or vascular occlusion; retinopathy due to prematurity/prematurebirth; infectious conditions, such as, e.g., CMV retinitis andtoxoplasmosis; inflammatory conditions, such as the uveitidies; tumors,such as retinoblastoma and ocular melanoma; and for the replacement ofinner retinal neurons, which are affected in ocular neuropathiesincluding glaucoma, traumatic optic neuropathy, and radiation opticneuropathy and retinopathy.

The treatments described herein can be used as stand alone therapies, orin conjunction with other therapeutic treatments. Such treatments caninclude the administration of a substance that stimulatesdifferentiation of the neuroretina-derived stem cells intophotoreceptors cells or other retinal cell types (e.g., bipolar cells,ganglion cells, horizontal cells, amacrine cells, Mueller cells).

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention as set forth in the appendedclaims. All publications, patents, and patent applications referencedherein are incorporated by reference in their entirety.

What is claimed is:
 1. A method of expanding multipotent human retinalprogenitor cells in vitro and maintaining the multipotency of said cellsin vivo, said method comprises the steps of establishing a cell culturecomprising isolated retinal multipotent progenitor cells, and culturingsaid cells under low oxygen conditions of from about 1% to about 6%oxygen content of the culture medium and for an effective amount of timewhile maintaining multipotency of said cells to provide an expandedpopulation of multipotent retinal progenitor cells, wherein said retinalprogenitor cells express both express both HIF1 alpha and HIF2 alpha. 2.The method of claim 1 further comprising isolating the expandedpopulation of cells from the culture media.
 3. The method of claim 2further comprising transplanting said cells into a host.
 4. The methodof claim 3 further comprising measuring said cells cultured under lowoxygen conditions for multipotency.
 5. The method of claim 1 furthercomprising culturing said cells in the presence of at least oneexogenous growth factor.
 6. The method of claim 1 wherein the effectiveamount of time comprises at least 10 passages in the low oxygenconditions.
 7. The method of claim 1 wherein the oxygen content of theculture medium is from about 2% to about 4%.
 8. The method of claim 1where the retinal progenitor cells are obtained from post-natal retinaltissue.
 9. The method of claim 1 wherein the retinal progenitor cellsare obtained from the retinal neurosphere.